Our accurate vision depends on the flow of visual information through precisely wired synaptic connections among axons and dendrites of retinal neurons with unique morphological and functional properties. In the vertebrate retina, each of the six neuronal cell types: ganglion, amacrine, bipolar, horizontal, and rod and cone photoreceptor cells are further divided into subtypes based on location, morphology and function. Of all retinal neurons, amacrine cells are the most diverse group with >30 subtypes being identified so far. They represent ~40% of neurons both in the inner nuclear layer (INL) and the ganglion cell layer (GCL), make up a majority of synapses in the inner plexiform layer (IPL), and contribute to a majority of visual processing in the retina. One of the key questions i how the many retinal neuronal subtypes are produced and wired during development. In this proposal, we focus on the amacrine cells associated with the sublaminar layer 3 (S3) of the IPL. The S3 sublamina separates the ON and OFF laminas in the IPL but its cellular makeup and function is poorly understood. Here, we have demonstrated LHX9, a LIM-homeodomain transcription factor, is expressed early in retinogenesis and its expression is tightly confined toa few amacrine cells in the INL and the GCL. In our preliminary study, we have shown that these LHX9+ cells are a subgroup of GABAergic amacrine cells and express GAD67 but not GAD65. LHX9-expressing cells are also LHX2-expressing subgroup of amacrine cells. Targeted deletion of Lhx9 in mice results in a nearly complete loss of these LHX2-expressing amacrine cells and strikingly, in the absence of the S3 sublamina, suggesting that LHX9 could be expressed in and be required for the development of a unique, S3-stratifying amacrine subtype cells. Interestingly, our preliminary data show that bNOS expression is significantly down-regulated in the Lhx9-null retina, suggesting a loss of bNOS-subtype of amacrine cells that are known to project in the S3 sublamina. Being a transcription factor with a known function in neuronal subtype development in the central nervous system, LHX9 likely plays a critic role in amacrine subtype specification and offers us a unique opportunity to ultimately elucidate the genetic pathway governing the formation of the S3 sublamina and its associated neural circuitry. In this proposal, we will fully characterize the subtype identity of these LHX9-expressing amacrine subtypes and will identify its circuitry within the retina. Second, we will analyze the retinal defects of Lhx9-null mutation, particularly the effect on amacrine subtype specification, differentiation of these Lhx9-expressing S3 stratifying amacrine cells, and the change in the functional properties and circuitry of the Lhx9-lineage cells. To elucidate the LHX9 regulatory pathway in the S3-stratifying amacrine cells, we will perform RNA-Seq of control and Lhx9-null retinas and use LHX9 ChIP-Seq to screen for downstream target genes of LHX9 and to identify the transcriptional network. Together, these studies will define the role of LHX9 in regulating the formation and neural circuitry of S3 sublamina and elucidate the transcriptional events that occur downstream of LHX9.